Project summary Medically important Acinetobacter species are opportunistic bacterial pathogens that can cause devastating disease in hospitalized patients and are responsible for ~2% of nosocomial infections in the US. These species are emerging as a public health threat worldwide due to their tendency to exhibit multidrug-, or even, pandrug-resistance. Therefore, we urgently need to develop new methods to treat and prevent MDR Acinetobacter infections. Critical for this advance is a detailed understanding of how these species cause human disease; however, much remains to be learned about the pathogenesis of Acinetobacter infections. Importantly, The Engel lab and others have identified three systems in Pseudomonas aeruginosa, another important opportunistic pathogen, that coordinately activate a virulence program in response to surface contact. These systems include the type IV pilus (TFP), the Chp chemosensory system, and the cAMP/Vfr axis. Together, these modules regulate twitching motility and expression of >200 virulence genes, such as the type II secretion system. It is intriguing that P. aeruginosa repurposed and combined together three distinct modules to regulate an acute virulence program when in other bacteria the TFP and the cAMP/Vfr axis control other processes. To test whether other bacteria utilize similar components and similar circuitry, the Engel lab performed a bioinformatics analysis to determine whether these modules were conserved and present together in other bacteria. Of high interest was the finding that medically important Acinetobacter species encode homologs of all three of these modules. Given the remarkable similarity in the lifestyles of Acinetobacter species and P. aeruginosa, including their ability to survive in diverse environments as well as to cause similar diseases in humans, I hypothesize that these three systems function coordinately to regulate the ability of a pathogenic Acinetobacter species to survive in diverse environments and to cause human disease. In Aim 1, I will use a combination of bacterial genetics, biochemical, and innovative microscopy assays to establish the functional outputs of the Acinetobacter Chp system and to determine if it is interconnected to the TFP and cAMP/Vfr modules. In Aim 2, I will make use of transcriptomic technologies to identify the virulence genes regulated by the Acinetobacter Vfr transcriptional regulator and to elucidate if Vfr activation depends on the TFP and Chp system modules. Taken together, these aims will allow me to determine whether the TFP, the Chp chemosensory system, and the cAMP/Vfr modules work together to regulate virulence in Acinetobacter species. These studies may identify new targets to develop novel antibiotics, and they might also provide a better understanding of the physiology and cell biology of medically important Acinetobacter species.